Cell culture models developed to study the action of tumor promoters are often limited by the requirement for serum in the culture medium. This problem can be overcome by the use of defined media selectively supplemented with highly purified and well-characterized attachment factors, growth factors and hormones. In this project a defined medium has been developed for the mouse epithelial cell line JB6 Clone 41, which has previously been shown to respond to tumor promoters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) in serum-containing media. In place of serum, the defined medium (referred to as 4F) contains fibronectin, insulin, transferrin and epidermal growth factor. JB6 Clone 41 was grown in 4F for over 35 generations without any noticeable change in cell behavior. In contrast, JB6 Clone 41 cells that had been induced to grow in soft agar by treatment with TPA were unable to grow in 4F, even though their growth in serum-containing media was unaffected. These results demonstrate that the use of defined media offers the advantage of exposing differences in the growth requirements of cells before and after treatment with tumor promoters.